Recent studies of reproductive frequency in the United States report that 7% of married couples (greater than 2 million couples) describe difficulty in achieving a pregnancy. See Fidler and Bernstein (1999) Public Health Reports 114:494-511. Many individuals now seek medical support for conception, including infertility diagnosis and assisted reproductive treatment. The monetary costs for such services are substantial, and financial commitment must increase to include pre- and post-natal care of multiple birth pregnancies often associated with infertility treatment. A tumult of legal and ethical issues have emerged regarding the rights of parents and unborn children that are conceived by an unconventional method. The escalating magnitude of monetary, legal, and ethical concerns when considering pregnancy has established infertility as a significant public health issue.
Rational treatment approaches for many andrological disorders resulting in infertility are still lacking. See Kamischke and Nieschlag (1999) Human Reproduction 14(Suppl. 1):1-23. The cause of male infertility is often unidentifiable, referred to generally as “idiopathic infertility”, or the presumed pathology is not yet met with an unequivocal therapy. Intracytoplasmic sperm injection has been a successful method for enabling fertilization in many cases, yet a less interventive treatment is still sought. The historical use of approaches now deemed ineffective emphasizes the importance of thorough studies during early stages of therapy development. In particular, there is a need for more sophisticated diagnostic tools that detect molecular bases of male infertility and for non-surgical therapies that are supported by solid physiological data. An initial effort in this regard is the development of animal models of male infertility.
A related medical incentive is the development of new methods for contraception. See Baird and Glasier (1999) BMJ 319:969-972. The prevalence of contraceptive use is increasing worldwide, however, existing contraceptive means are limited by adverse side effects, inconvenience, and remaining instances of ineffectiveness. In particular, there are presently no safe and reversible means for male contraception. One strategy that has been explored recently is an immunological approach for disrupting endocrine or physiological events that normally promote pregnancy. Vaccines that comprise antigens of sperm plasma membrane proteins, zona pellucida proteins of the egg, or gonadotropin releasing hormone have shown success in suppressing fertility when administered to several mammalian subjects, including humans. See U.S. Pat. Nos. 6,096,318 and 6,132,270; Barber and Fayrer-Hosken (2000) J Reprod Immunol 46:103-124; Paterson et al. (2000) Cells Tissues Organs 166:228-232; Srivastav (2000) J Reprod Fertil 119:241-252; Feng et al. (1999) J Reprod Med 44:759-765; Naz (1999) Immunol Rev 171:193-202; Talwar (1999) Immunol Rev 171:173-192.
Although animal use and clinical trials of immunocontraceptive vaccines are encouraging, existing vaccines present significant complications, for example, auto-immune reactions. Thus, current research is focused on identifying new antigens that can provide safer vaccines. Animal models with implications for post-testicular human male contraceptives acting at the epididymis offer promising leads. See Nikkanen et al. (2000) Contraception 61:401-406; Cooper and Yeung (1999) Hum Reprod Update 5:141-152. To establish molecular targets for vaccine and drug development, proteins that are essential for epididymal function have been identified (Srivastav, 2000; Diekman et al. (1999) Immunol Rev 171:203-211; Costa et al. (1997) Biol Reprod 56:985-990; Sonnenberg-Riethmacher et al. (1996) Genes Dev 10:1184-1193).
During their transit through the epididymis, spermatozoa undergo biochemical and morphological changes to acquire motility and the ability to fertilize an oocyte in vivo. The maturation process occurs progressively along the epididymal duct and is believed to depend on epididymal secretory proteins. The epididymal epithelial cells secrete proteins in a highly regulated and regionalized manner such that spermatozoa encounter luminal fluid protein in a specific sequence. Indeed, each region within the epididymis is a unique microenvironment adapted with a characteristic milieu of ions, organic solutes, proteins, and steroids. See Cornwall et al. (2001) in “The Epididymis”, Plenum Press. Spatially-restricted gene expression as well as regional differences in cellular morphology define three distinctive regions of the epididymis known as the caput, corpus, and cauda. These structural subdivisions and highly regionalized gene expression therein are observed in the epididymis of several organisms, including humans (Krull et al. (1993) Mol Reprod Dev 34:16-34).
Regionalization of the epididymis likely fulfills an essential and cumulative role in the maturation and survival of spermatozoa. In support thereof, targeted mutation of the mouse c-ros tyrosine kinase receptor confers male sterility, although sperm production is not affected. c-ros is normally expressed in the initial segment of the epididymis, and animals lacking c-ros function show specific underdevelopment and lack of cellular differentiation within the initial segment (Sonnenberg-Riethmacher et al., 1996). Sperm taken from a c-ros mutant mouse are less motile due to flagellar angulation, suggesting that failure of differentiation of epithelial cells in one segment of the epididymis can affect sperm maturation and survival (Yeung et al. (1999) Biol Reprod 61:1062-1069).
To generate regionalization within the epididymal epithelium, gene expression is precisely controlled, in part through transcriptional regulation. Transcription factors modulate transcription by binding DNA cis-regulatory sequences, most often located upstream of the gene promoter and transcription start site, and by concomitantly affecting assembly of cellular transcriptional machinery at the relevant promoter. Therefore, cis-regulatory sequences of epididymal-specific genes and the transcription factors that are operative through these sites are important elements in understanding epididymal function as it contributes to sperm maturation. Current approaches to identify mechanisms involved in region-specific gene expression in the epididymis have been limited by a lack of identified transcriptional regulatory proteins which are key to this process. See Cornwall et al. (2001).
Candidate regulators include components of retinoid signaling pathways. Most elements known to be involved in retinoid signaling are present in the epididymis, including epididymal retinoic acid binding protein (mE-RABP), cellular retinol-binding protein type I (CRBP I), cellular retinoic acid binding protein type I (CRABP I), retinoic acid receptor alpha (RARα), retinoic acid, and retinyl esters. Moreover, studies addressing the function of such elements emphasize the important role of retinoid signaling pathways in epididymal integrity. In retinoid deficient animals, there is widespread squamous metaplasia and keratinization of the epididymal epithelium (Wolbach (1925) J Exp Med 42:753-777), and abnormal synthesis and secretion of several epididymal proteins (Astraudo et al. (1995) Arch Androl 35:247-259). Similarly, overexpression of a dominant negative form of RARα leads to disorganization of the epididymal epithelium and concomitant infertility (Costa et al., 1997). In a related study, RARα knockout mice display aspermatogenesis and vacuolization of the epididymal epithelium (Lufkin et al. (1993) Proc Natl Acad Sci USA 90:7225-7229), and animals lacking both RARα and RARγ function show epididymal dysplasia (Mendelsohn et al. (1994) Development 120:2749-2771).
The mE-RABP protein is of particular interest among regulators of retinoid signaling, as it appears to be expressed selectively in the mid and distal caput of the epididymis. mE-RABP is a member of a family of secreted lipocalin proteins. Structural analyses reveal that lipocalins comprise an eight-stranded β barrel that is closed at one end by an α-helical turn, thereby forming a hydrophobic binding cavity. This hydrophobic pocket is well-adapted for noncovalent binding and transport of small lipophilic ligands. mE-RABP binds active retinoids (9-cis and all-trans retinoic acid), and functions as a retinoid carrier protein in the epididymis. See Ong et al. (2000) Biochim Biophys Acta 1482(1-2):209-17.
Recent studies by the co-inventors of the present application have identified a similar gene encoding a 17 kDa lipocalin, Mouse Epididymal Protein of 17 kDa (mEP17) (Lareyre et al. (2001) Endocrinology 142:1296-1306). mEP17 and mE-RABP are significantly related by several measures that collectively suggest mEP17 also functions as a regulator of retinoid signaling in the epididymis. First, mE-RABP and mEP17 are positioned adjacent to each other on mouse chromosome 2. Exon/intron boundaries are strictly conserved between mE-RABP and mEP17, supporting that these genes arose by gene duplication. Second, mEP17 shows regionalized expression in the epididymis. mEP17 expression is limited to the initial segment of the caput epididymis, while mE-RABP is expressed in the adjacent mid and distal caput epididymis. Third, the mEP17 protein contains two motifs (G-X-W and T-D-Y) and two cysteine residues that are characteristic features shared by members of the lipocalin protein family. With the exception of these motifs, mEP17 shows low sequence similarity with other known lipocalins. However, it is well established that lipocalin family members do not show significant sequence homology (average 25% identity and 50% homology between representative members). Rather, lipocalins are more clearly related by assessing homology of secondary and tertiary structure. The tryptophan residue of the G-X-W motif is required for binding of lipophilic ligands, and the two cysteine residues form a intramolecular disulfide bond that influences ligand affinity. In addition, a putative signal sequence at the amino-terminal of the mEP17 precursor suggests that it is cleaved to generate a mature secreted protein, consistent with its identification as a lipocalin. These structural similarities between mEP17 and other lipocalins, most significantly mE-RABP, suggest that mEP17 is also a carrier for retinoid ligands.
The present invention relates to a current challenge in developing animal models of infertility, male fertility treatments, and male contraceptives. To this end, the present invention provides an isolated promoter region of the mEP17 gene, an isolated nucleic acid molecule encoding a human mEP17 gene (hEP17), an isolated promoter region of hEP17, and chimeric genes comprising the disclosed sequences. Host cells expressing a recombinant EP17 gene or an mEP17 promoter region operably linked to a reporter gene sequence are useful in screening assays for discovery of substances that modulate EP17. A chimeric gene comprising an mEP17 promoter region can also be used to direct transcription of a heterologous nucleotide sequence in the epididymis of a host organism. The present invention further provides an EP17 polypeptide that can be used for vaccine or drug development. By provision of epididymal lipocalin nucleotide and polypeptide sequences, and methods for using the same, the present invention meets a long-felt need for advancement in fertility research.